Method for the selective performance of thin-layer chromatography and an apparatus for performing the same



June 15, 1965 M. BRENNER -:-AL 3.189,54!

HETHOD FOR THE SELECTIVE PERFORMANCE OF THIN-LAY CHROHATOGRAPHY AND AN APPARATUS FOR vPERFORMING Fea ay e, 1962 THE SAIIE 3 Shets-Sheet l June 15 1955 M. BRENNER E-rA. 3,189,541

ETHOD FOR THE SELECTIVE PERFORMANCE OF THIN-LYER CHROTOGRPHY AND AN PPARTUS FOR PERFQRMNG' THE SME V Filed lay 9, 1962 3 Sheets-Sheet 2 MAX BRENNER Am ALos- Nve/?mesala Iuvamzs Arrakufrs June. 15, 1965 M. BRENNER ETAL IETHOD FOR THE SELECTIVE PERFORMANCE OF THIN-LAYER' CHROATOGRPHY AND AN APPARATUS FOR PERFORMING THE SAIE 3 Sheets-Sheet 3 Filed lay 9, 1962 Rf DEMME Mmmwm www. u EE RI BMW XS AM MM United States Patent O Basel, Switzerland Filed May 9, 1962, Ser. No. 193,435 Claims priority, application Switzerland, May 10, 1961, 5,607 61 14 Claims. Cl. 210-31) A very modem 'form lof chromatography is the socalled thin-layer chromatography. The said name is based on the use of the adsorption agent in the form of a thin layer on an inert base. This arrangement possesses a number of known advantages as compared to the conventional adsorption column. According to the standard method the distance to be traversed by the solvent used as mobile phase for the chromatographio separation is limited by the dimensions of the apparatus. While this is of no significance for the separation of substance mixtures, the components of which have distinctly different migration velocities, the separation of substances having very similar migration velocities is only possible if a correspondingly large path of migration would be provided. 'Since the path of migration is always ,smaller than the distance to be traversed by the solvent, the separation of substances with very similar migration velocities thus requires' a large distance to be traversed by the mobile phase and this requirement in its tum gives rise to the requirement for large, even very large apparatus dimensions. In paper chromatography, a known operating technique closely related to the said thin-layer chromatography, the above said disadvantage may -be avoided, on the one hand by selecting the mobile phase e.g. such that it moves at least twice as quickly as the quickest component of the mixture of substances and on the other hand by virtually increasing the distance to be traversed by said mobile phase, that is to say allowing the mobile phase to fiow beyond the area of the adsorption agent. This latter effect is most simply attained by a vertical arrangement of the paper, by a descending fiow direction of the mobile phase and by dripping ot from the lower ledge of the paper. The solvent being the mobile phase' is thus allowed to fiow through the adsorption agent, the use of this arrangement being known as run-through chromatography; run-through method and run-through principle are synonymous expressions. I

As known, the run-through principle considerably broadens the range of use of paper chromatography. An arrangement for the thin-layer chromatography analogous to the run-through paper chromatography is indeed conceivable, but Complicated, and has thus not previously been described. It is, however, evident that a further improvement of the in fact already excellent separation chances of the thin-layer chromatography by means of a suitable arrangement for use of the run-through principle must further considerably increase the great practical advantage of thin-layer chromatography.

One of the objects of the present invention is a method for the selective performance of thin-layer chromatography 3,89',54l Patented June 15, 1965 according to the run-through method or standard method, while another object of the present invention is an apparatus for performing the said new method. The method for thin-layer chromatography is performed in that mixtures of substances to be separated are applied along a starting line S on the thin layer, the solvent is allowed to fiow from a solvent container B through a fluid bridge F onto the .covered thin layer, the same is allowed to fiow through this latter and, if desired, to fiow out of the covered thin layer for the time required for further substance separation, whereupon the solvent can evaporate or drip off.

The apparatus which is surprisingly simple is characterized by that the thin layer used for the separation of the substances lies on a carrier plate T, is sealed off from the surrounding area by a cover plate D positioned parallel to the said carrier plate and `by support or guide members positioned on two sides thereof, its front end being connected with a Container B for the solvent by means of a fluid .bridge F covered by the carrier plate and the end portion opposed to the front end being open or connected with the surrounding area by means of a fluid bridge.

When using the new apparatus of the present invention for run-through thin-layer chromatography, the

proven rules for thin-layer and paper chromatography, well-known to the man skilled in the art, are advantageously followed with respect to the choice of adsorption agent and solvent, as well as the chromatogram loading. It is thus obvious that to separate substances which behave chromatographically very similarly, solvent systems will preferably be used, in which the substances yto be 'separated move very slowly, whencompared with thel speed with which the solvent, i.e., the mobile phase, is

moving.

Filter paper, for example, can be used as fluid bridge. It is, however, also possible to use another absorbent material or a narrow slit. The transport of the liquid fluid may take place by capillary forces, by gravity, centrifugal force or pressure. The solvent is thus, by way of example, transferred by the capillarity of the fluid bridge onto the thin layer and after fiowing through the migration path, i.e., the portion of the thin layer covered by the cover plate, can evaporate or drip off, whereby this can take place eitherdirectly or across a fluid bridge, c g., a filter paper adjacent the layer, which projects into the surrounding area. The apparatus is particularly simple with the thin layer in horizontal position and with evaporation from its uncovered, open end portion as shown in FIG. 5 of the enclosed drawings. v

The support or guide members mounted longitudinally between the carrier and cover plates serve for Vsealing and must, more particularly at the junction of the fluid bridge with the layer, guarantee sufficent spacing between thev layer and the cover plate to hinder solvent from fiowing into the intermediate area by reason of capillarity. It .is constructionally particularly simple to mount the cover plate on runners which project beyond the upper edge of the layer by 0.01 to 10 mm. A further appropriate solution consists in fixing the desired space between the cover plate and the carrier plate or thin layer by a guide rail.

vhereby of no significance.

The fluid Container, carrier plate, cover .plate and support or guide members are advantageously made of `a chemically inert material, such as glass, metal, a vsynthetie substance or metallized synthetic substance; if desired, a combination of these su-bstances may also be used.

The use of the new apparatus of the present invention is, however, not only restricted to cases in which Vone must use the runthrough principleto attain separatOn. On the contrary, it is characteristic for the novel arrangement that it can generally also be employed wherever it sufices to use the usual thin-layer chromatographic standard device described by Stahl, Archiv der Pharmaziej' 292/64, 411 (1959). In such a lcase one can proceed by interrupting the chrornatography when the solvent front has run through approximately the path from the starting 'line to the end of the cover plate (standard method).

It is thus a particular characteristic of the said apparatus for thin-layer chromatography that it can be employed both in the run-through method and in the standard method. The chamber saturation which is so important according to the usual standardized apparatus of Stahl is The present invention thus excellently combines simplicity of apparatus with multiplicity of use. The individual parts, the assembly, the process and, by means 'of examples, the use of an apparatus according to the invention will bedescribed hereinafter, .without in any way thereby lirniting .the nature, scope or application range vof the invention. The enclosed drawings help Vto illustrate the invention: FIG. l: Container B made of V4A steel for the mobile phase; in perspective view;

V FIG. la is a sectional view of the container of FIG. l

FIG. 2: Cut sheet for the filter paper F;

FTG. 3: Carrier plate T made of glass with thin-layer and bare edges;

FIG.'4: Cover plate D made of glass with runners and mounted lilter paper F in perspective view;

FIG. 4a is a sectional view of one edge of the cover plate of FIG. 4; FIG. Apparatusgfor thin-layer chromatography with selective use of the run-through or standard method in section; and

FIG. Sa is a perspective view of the apparatus of FIG. 5.

ABBREIATIONS CITED IN THE DRAWINGS A. Individual .parts of the flpparatus (l) Container B made o sta'nlesssteel- (FIG. 1).- It has a plane cut upper surface and serves as receptacle for the mobile phase. Two short polyethylene tubes R are inserted through two holes L in the side wall and bent upwards according to FIG. l (cross-Section). The mobile phase will later be poured in through one. of said tubes, the other one servng for pressure equalization (see below).

. t 4 (2) `Filter'paperF (FIG. 2). It -transfers vthe mobile phase onto the layer. The .part egh is -adapted to the inner profile of Vthe container.

(3) VCarrier plate T made of glass compr's'ng the thinlayer (FIG. 3).-0n two opposed edgessuch an amount of the layer is wiped off -that the runners (see below) just fit on the bare strips. mm. away from the front plate edge -vis Vthe starting line S where the substances are :to be applied (FIG. 3).

(4) Cover plate made of glass w''th runners G v(FIG. 4).. Two plane-cut glass bands welded longitudinally onto the cover plate serve as runners. The height of the runners permits selective adjustment of the space between the -layerand the cover plate; simultaneously, .the runners act as sealing.

(5) Six strong paper clips K. They .serve to press the Container B and the carrier plate T against the cover plate D (FIG'. 5). l

(6) Support U The same can have ring form, a cork ring, for example, being suitable k(cf. FIGS. 4 and 5).

B. Assembly and method The cover .plate with the runners facing upwards-is laid on lthe cork ring U, the filter paper is folded along the t paper should not touch the runners and theedge ab should be slightly spaced away from the-front edge of the cover plate (1-3 mm.). This position is provisionally fixed with a paper clip. The carrier plate with the layer and the substances to be chromatographed, which latter have already previous to assembly been applied onpoints along the starting line S (FIG. 3), is now laid onto the cover plate in such a way that the bare strps come to rest on the runners and the front edge of the carrier vplate exactly corresponds with the front edges of the runners. The carrier plate and the cover plate are nowpressed together by four paper clips. and the clip on the filter paper,

which has now become superfluous, is removed. TheA upper part of the filter paper is bent along .the connecting line (FIG. 4) into verticalposition and the clipped together-plates arevinverted so that the filter paper is now on the underside. As shown in FIG. 5, the container B is clipped with two clips K under the cover plate in :such mannerthat the vertically upright part of the filter paper reaches to the bottom of -B and the horizontal part' acts as sealing between the cover plate and. the upper surface of` the container B. The container should lieclose against the carrier plate. Mobile phase is poured in through a polyethylene tube,a momentary uniform wettingof the filter paper-is. attained by slighttilting andk the. .arr'angei ment can now be left alone. The mobile phase contain'- ng the solvent evaporates afterrunning through the layer protected by the cover plate on the still remaining strip4 (FIG. 5) sutficiently quickly to keep the ,chromatographyl as long as desired in action. If required, mobile phase"- is to be refilled intocontainer B. 3

Distrrbances..-Varations of room temperature cari sometimes (particularly with severe flow of air). lead, toV

the mobile phaseV condensing on the cover plate. Insuch by. layingv So.-calledl edge 'effects' cases it suffices to warm the cover plate slightly oneishand on it for ashort time. are avoidable with careful assembly.

C. Use

The arrangement described can, according to'theinven-f tion, be used to separate dilficultly and readily separable' substance mixtures. The following table gives a number of cxamples and the conditions observed for scparation. The adso'ption agent vis in :ll cases silicic gclfor thinlayer chrornatog'zuns according to Sthl (Mcrck); the

space between the starting line and the end of the cover plate is 16.5 cm., the height of the runners is l mm.

TABLE Re- Ex- Snbstincc mixture Solvcnt system qlrud nnple. time lu N o. hours 1 Isoleuclne lcu- Methyl ethyl 5 clne. ketone/pyridinel water 15:15: 2 v./v.). 2 N-(z-i-dinitro- Benzene/pyri- '2-3 phenyD-derivadine/glaral tives ot isoleuaeetic acid eine leucine -l- (80:20:15 v./v.). Norleucine. Run-through 3 N-(2z4-dinitrodo 2-3 method. phenyD-deriva- V tives of norvaline valine. 4 N:N'-di-(2:4- Chloroform/meth- 2-3 dinitrophenyD- annl/glacal lysine -1- OzN- acetic acid di-(2z4-dnitr0- (95:5:1 v.lv.). phenyD-tyrosine. 5 Arginine hydron-Propanol/water 1-2 chloride (70:30 v./v.). glycine -1- histidinehydrochloride phenyl alanine valine.

6 N-(2z4-dinitro- Clloroform/ 1 Standard phenyD-derivahenzyl alcohol l method. tives ol' alunine glacial acetic a-amino buacid (7023013 tyrie acid v./v.). a-nmino mprylic acid giycinc scrine. 7 Dimethyl/yellow Bcnzenc 0.25

+ indophenol sudan red G.

What we claim is:

1. A method for the selective performance of thin-layer chromatography which comprises applying a mixture of substances to be separated along a starting line on a thinlayer, causing a solvent to flow from a solvent container through a fiuid bridge onto the covered thin-layer, allowing the solvent to flow through said layer for the time required for substance separation.

2. A method for the selective performance of thinlayer chromatography which comprises applying a mixture of substances to be separated along a starting line on a thin-layer, causing a solvent to flow from a solvent container througha fiuid bridge onto the covered thinlayer, allowing the same to flow through said layer and out of the covered thin-layer for the time required for further substance separation, an evaporation step being finally effected.

3. A method for the selective performance of thinlayer chromatography which comprises applying a mixture of substances to be separated along a starting line on a thin-layer, causing a solvent to flow from a solvent container through a fiuid bridge onto the covered thin-layer, allowing the same to flow through said layer and out of the covered thin-layer for the time required for furthcr substance separation, a dripping-ofi' step being finally g etfected.

4. A method for the selective performance of thinlayer chromatography which comprises applying a mixture of substances along a starting line on a thin-layer, causing a solvent to flow from a solvent container through a fiuid bridge onto the covered thin-layer, allowing the 6 lf; and separation of the components of the mixture is thus effected, which apparatus comprises a carrier plate bearing a thin-layer used for the separation; a container for the solvent; a fiuid bridge constitutng means for transporting the solvent from the solvent container to one end of the thi-layer; and a cover plate constituting means for isolating the thin-layer from the outside environment.

6. An apparatus as in claim 5, in whichthe end of the thin-layer opposite the fiuid bridge connecting the solvent and thin-layer is connected to the surrounding environment through a second fiuid bridge.

7. An apparatus according to claim 5, in which the fiuid bridge constituting means for transporting the solvent from the solvent container to one end of the thin-layer is covered by the cover plate.

8. An apparatus as in claim 5, in which the container for the solvent is covered by the cover plate.

9. An apparatus as in claim 6, in which the fiuid bridges and the container for the solvent are covered by the cover plate.

10. An apparatus as in claim 5, containing means for introducing the solvent into the solvent container from the outside area.

11. An apparatus as in claim 5,' in which the cover plate has runners firmly attached to the bottom at the outer edges thereof, said runners having a thickness equal to or slightly less than the thickness of the said fiuid bridge, which runners serve as support and guide layer from the outside environment.

12. An apparatus according to claim 5, in which the fiuid bridge comprises filter paper.

13. An apparatus according to claim 5, in which the container for the solvent, thefluid bridge, and the carrier plate are pressed against the cover plate.

14. An apparatus for the performance of thin-layer chromatography in which a solvent is flOwed through a thin-layer to which a mixture has been applied and separation of the components of the mixture is thus effected, which apparatus comprises a carrier plate bearing a thin-layer for the'said separation; a container for the said solvent, said container being positioned adjacent to one end of the said carrier plate; a fiuid bridge extending between the said solvent container and the solvent container end of the said thin-layer and constituting means for transporting the solvent from the solvent container to the said thin-layer; a cover plate having firmly attached to the bottom at the outer side edges thereof runners, said runners having a thickness equal to or slightly less than the thickness of the said fiuid bridge, which runners serve as support and guide members as well as means for isolating the said thin-layer from the outside environment; said cover plate covering the container for the solvent, the fiuid bridge and the carrier plate and constituting means for isolating from the surrounding environment the container for the solf vent, the fiuid bridge and the thin-layer; means for firmly' pressing the cover plate against the container for the solvent and for pressing the runners of the cover plate against the outer side edges of the thin-layer side of the said carrier plate; and means for introducing the solvent into the solvent container and equalizing'the pressure in the solvent container when the cover is in position. 

1. A METHOD FOR THE SELECTIVE PERFORMANCE OF THIN-LAYER CHROMATOGRAPHY WHICH COMPRISES APPLYING A MIXTURE OF SUBSTANCES TO BE SEPARATED ALONG A STARTING LINE ON A THINLAYER, CAUSING A SOLVENT TO FLOW FROM A SOLVENT CONTAINER THROUGH A FLUID BRIDGE ONTO THE COVERED THIN-LAYER, ALLOWING THE SOLVENT TO FLOW THROUGH SAID LAYER FOR THE TIME REQUIRED FOR SUBSTANCE SEPARATION.
 5. AN APPARATUS FOR THE PERFORMANCE OF THIN-LAYER CHROMATOGRAPHY IN WHICH A SOLVENT IS FLOWED THROUGH A THINLAYER TO WHICH A MIXTURE OF SUBSTANCES HAS BEEN APPLIED AND SEPARATION OF THE COMPONENTS OF THE MIXTURE IS THUS EFFECTED, WHICH APPARATUS COMPRISES A CARRIER PLATE BEARING A THIN-LAYER USED FOR THE SEPARATION; A CONTAINER FOR THE SOLVENT; A FLUID BRIDGE CONSTITUTING MEANS FOR TRANSPORTING THE SOLVENT FROM THE SOLVENT CONTAINER TO ONE END OF THE THIN-LAYER; AND A COVER PLATE CONSTITUTING MEANS FOR ISOLATING THE THIN-LAYER FROM THE OUTSIDE ENVIRONMENT. 